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ORIGINAL ARTICLE
Year : 2013  |  Volume : 3  |  Issue : 3  |  Page : 282-288

Laboratory evaluation of cases of meningitis attending a tertiary care hospital in India: An observational study


1 Department of Microbiology, College of Medicine and Sagore Dutta Hospital, Katihar Medical College and Hospital, West Bengal, India
2 Department of Pathology, College of Medicine and Sagore Dutta Hospital, Katihar Medical College and Hospital, West Bengal, India

Date of Submission08-Jul-2012
Date of Acceptance23-Dec-2012
Date of Web Publication10-Jul-2013

Correspondence Address:
Moumita Adhikary
231/1, R.B.C. Road, P.O: Naihati, North 24 Parganas, West Bengal - 743 165
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.114861

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   Abstract 

Context: Diagnosis of infection involving the central nervous system is of critical importance. Aims, Settings and Design: Objectives were to isolate and identify the organisms causing bacterial meningitis in patients admitted in the intensive care unit of a tertiary care hospital in Bihar, India. To assess the clinical presentation and to further determine the isolates causing meningitis by antigen testing. Materials and Methods: Cerebrospinal fluid (CSF) and blood samples were collected from 83 patients with signs and symptoms of meningitis and were cultured on 5% sheep blood agar, MacConkey's agar and chocolate agar. Smears were made with the centrifuged deposit of CSF and stained by Gram stain, Ziehl Neelsen stain and negative staining. The supernatant was used for latex agglutination test (LAT). Antibiotics susceptibility tests were also performed for all isolates. Statistical Analysis Used: S.S.P.E version 11. Results: Most of the cases of meningitis occurred in winter and spring. Out of 83 cases 38 (45.8%) were found to be cases of bacterial meningitis. The most common presenting feature in both bacterial and aseptic meningitis was fever, followed by altered sensorium. Only 15.7% (13/83) of cases had neck rigidity. The organisms isolated were Escherichia coli 25.6% (10/39), Staphylococcus aureus 15.4% (6/39), Streptococcus pneumonia 10.2% (4/39), Klebsiella species 10.2% (4/39) and Pseudomonas areuginosa 10.2% (4/39). LAT detected bacterial antigens in 16.9% (14/38) cases. The common etiological agent identified was E. coli 10/38 (71.4%) and followed by Streptococcus pneumonia 4/38 (28.6%). Conclusions: To conclude, Gram negative bacilli were found to be more frequently isolated in our series of patients with E. coli being the most common isolate. Established organisms of meningitis such as Haemophilus influenzae and Niesseria meningitidis were not isolated at all, despite all our efforts.

Keywords: Acute bacterial meningitis, Cerebrospinal fluid, Latex agglutination test


How to cite this article:
Adhikary M, Chatterjee RN. Laboratory evaluation of cases of meningitis attending a tertiary care hospital in India: An observational study. Int J Nutr Pharmacol Neurol Dis 2013;3:282-8

How to cite this URL:
Adhikary M, Chatterjee RN. Laboratory evaluation of cases of meningitis attending a tertiary care hospital in India: An observational study. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2020 Apr 3];3:282-8. Available from: http://www.ijnpnd.com/text.asp?2013/3/3/282/114861


   Introduction Top


Meningitis is one of the medical emergencies related to infectious diseases, which is potentially associated with a high rate of complications. [1] Meningitis is the inflammation of the meninges, a protective membrane that surrounds the brain and the spinal cord. However, this inflammation not only involves the meninges but also spreads to the subarachnoid space and to the cerebrospinal fluid (CSF) contained within it. [2]

The patient presents with fever, stiff neck, headache, nausea, vomiting, lethargy, anorexia, irritability, photophobia and positive Kernig's sign. [3] Bacterial meningitis is mainly caused by Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae and enteric Gram negative bacteria. [4]

The incidence of meningitis in U.S., between 1978 and 1981, was approximately 30 per million population, which dramatically reduced in 1995 due to vaccination with Hib and pneumococcal vaccines. [5] The picture was quite different in the U.K. where there was an annual incidence rate of 16 per 1,00,000 children aged 0-16 years. [6] In India, Ahmedabad, the incidence varied from 12.12% to 56% [7] whereas another study found only 1.5% incidence of meningitis out of all admissions in the hospital. [8] In a study carried out in Maharashtra, India, 36% of the cases were diagnosed as bacterial meningitis. [9] Another 10 year retrospective study in South India showed the incidence to be 73.8%. [10]

Meningitis may be associated with significant mortality even after institution of therapy. [3] Nearly one in four adults with acute bacterial meningitis (ABM) will die and many survivors sustain neurological deficit. [11] Early implementation of appropriate antimicrobial therapy requires prompt identification of the infecting pathogen. The non-specific clinical presentation and lack of laboratory facilities can delay or obscure diagnosis. [12]

Although culture is considered to be the gold standard, [13] CSF turbidity, bacteria on direct Gram stained preparation [14] and pleocytosis, [15] increase in protein and decrease in sugar are preliminary indicators. However, culture for fastidious organism is difficult and time consuming and produce false negative results; [16] hence the detection of soluble antigens in CSF in suspected cases by the latex particle agglutination test is considered an important diagnostic tool which has a high sensitivity, specificity, simplicity in execution, rapidity, and interpretation. [16]

To reduce the mortality and morbidity owing to meningitis, an early establishment of the diagnosis and early institution of therapy are imperative. [17] This study intends to identify the magnitude of the disease in patients attending Katihar Medical College, Bihar, with respect to seasonal variation, age, and sex with the identification of the organisms involved and detection of antigens.


   Materials and Methods Top


The study was conducted in the Department of Microbiology between November 2008 and 2010. All patients admitted with clinical suspicion of meningitis in the Medicine and Pediatric ward were included. Demographic details including geographic location, age, and sex were collected. A detailed history including prior antibiotic intake was recorded in a standard proforma. A total of 83 blood and CSF samples were collected. Patients unfit for lumbar puncture were excluded from the study.

CSF procedures performed included total and differential white blood cells (WBC) count, protein and glucose concentration, Gram stain, culture, antibiotic sensitivity tests, and LATs. Blood culture was carried out in each of the cases. The CSF was collected under aseptic conditions, each containing 2 ml of fluid, and brought to the central laboratory immediately. Macroscopic appearance was noted. The first vial was used for bacteriological examination, the second was used for cytological examination, and the third for biochemical examination.

Apart from CSF, 5-10 ml of blood in adults and 3-5 ml of blood in cases of neonates and children were collected immediately before institution of antibiotics.

CSF specimens were prepared for microscopic examination by centrifugation at 1500 rpm for 15 min at room temperature. After conventional Gram staining slides were examined by light microscopy at ×40 magnification to allow quantification of WBC and then at ×100 magnification under oil immersion. The presence of organisms and their morphologies were reported. Besides Gram stain, Ziehl Neelsen and nigrosin staining were also carried out to identify acid fast bacilli and fungus, respectively.

For culture, approximately 0.15 ml of the deposit of the centrifuged CSF specimen were inoculated onto plates of 5% sheep blood agar, chocolate agar, and Mac Conkeys' agar medium. The deposit was also inoculated in 3-4 ml of brain heart infusion broth. The blood agar plates were streaked with Staphylococcus aureus (Plazens) strain to detect Haemophilus influenzae. Bacitracin 0.04 μl and optochin discs were placed at 37°C in 5-10% CO 2 in a candle jar and examined daily for 3 days. A slope of Lowenstein-Jensen medium was also inoculated for each specimen and incubated in a separate incubator at 37°C for 8 weeks. The slopes were examined on the 4 th day to identify any obvious contamination and thereafter once a week for up to 8 weeks. Two slopes of Sabouraud's Dextrose agar were inoculated and 1 tube was incubated at 37°C and the other at 25°C for 4 weeks. They were examined once a week for up to 4 weeks. Broth cultures were incubated at 37°C and examined daily for 7 days. Blood culture bottles were also incubated at 37°C for 7 days. Turbidity was checked everyday up to 7 days and irrespective of turbidity, subcultures were carried out. Any growth present on the plates was identified approximately according to the standard recommendations. If the broth culture displayed turbidity, a repeat smear was performed for Gram staining and subcultures.

CSF was tested for bacterial antigens by latex agglutination test using the BIO-RAD PASTOREX MENINGITIS Kit. The kit was provided with sera to detect antigens of N. meningitidis A/B/C/Y/W135, E. coli K1, H. influenzae type B, Strep. pneumoniae, Streptococcus group B. The test was carried out with a supernatant according to the manufacturer's instructions. Quality control was monitored throughout the laboratory work.

Statistical analysis was carried out using the SPSS (version 11.5) software for CSF cell count, glucose levels, and protein content.


   Results Top


The study population included 83 patients clinically diagnosed with meningitis, of which 50 (60.2%) were male and 33 (39.8%) female, between all age groups. Maximum number of patients belonged to (30 [36.1%]) 18-65 years, and the least (11 [13.6%]) was found from birth to 6 months.

The presenting features included fever in all 83 cases (100%), altered sensorium in 63 (75.9%), and inability to take food in 62 (74.6%); lethargy, vomiting, and neck rigidity were present in 35 (42.2%), 26 (31.3%), and 13 (15.7%) patients, respectively [Table 1]. The overall maximum number of cases of meningitis was recorded during April 16 (19.3%), March 9 (10.8%), and September 8 (9.6%), whereas cases of bacterial meningitis occurred more frequently between January and April 22 (57.9%) [Figure 1].
Figure 1: Month wise distribution of meningitis

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Table 1: Signs and symptoms in cases of meningitis

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The overall macroscopic appearance of CSF samples was clear in 43 (51.8%) cases and, cloudy in 29 (34.9%). Cases of bacterial meningitis had turbid or cloudy appearance in 28 (73.6%) instances [Table 2]. Cell count was high in bacterial meningitis 23 (60.5%) cases, which was significantly higher than aseptic meningitis (P0 = 0.000) [Table 3]. Sugar was very low (10-30 mg/dl) in 21 (55.3%) cases which was significantly lower than aseptic meningitis (P = 0.034) [Table 4]. However, the protein count of CSF was significantly higher (89.5% 101-500 mg/dl) than that of aseptic meningitis (P0 = 0.000) [Table 5].
Table 2: Macroscopic appearance in CSF specimens of meningitis

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Table 3: CSF cell count among cases of meningitis

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Table 4: CSF glucose among cases of meningitis

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Table 5: CSF protein among cases of meningitis

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The CSF culture was positive only in 38 (45.7%) cases. Both the organisms Citrobacter sp. and Provindencia sp. were isolated from the CSF of a single patient with ruptured meningocele. The total number of isolates was 39 out of 83. E. coli was the most common pathogen isolated (10 [25.6%]) followed by S. aureus (6 [15.4%]). Pseudomonas areuginosa, Klebsiella sp., and Strep pneumoniae were present in 4 (10.3%) cases each. Mycobacterium tuberculosis was isolated from only one patient [Table 6], [Figure 2]. The corresponding blood cultures were positive in 20 (52.6%) cases. Of the 38 samples, Gram stain of CSF produced positive results in 33 (86.8%) cases. Of the remaining 45 samples, 3 (6.6%) were believed to yield a misleading false-positive result on Gram stain, wherein the culture yielded no growth and aerobic spore bearers and no pus cells were found on direct examination. Of the remaining 42 samples that were negative for microscopic examination, in 4 (8.8%) samples the culture yielded organisms that were proved as contaminants (aerobic spore bearers). Zeihl-Neelsen staining was positive in only one case where the CSF sample had a cobweb formation macroscopically. Organisms such as Streptococcus agalactiae could not be recovered from the blood samples [Table 7].

LAT detected bacterial antigens in 14 (16.8%) out of the 38 culture positive cases. The common etiological agents identified were E. coli (10 [71.4%]) and next were Streptococcus pneumoniae 4 (28.5%). In the remaining 24 (63.1%) cases, no organisms could be detected. This is because of the unavailability of the antigens to some organisms. [Table 8] shows the correlation of culture with latex agglutination for the CSF specimens along with Gram stain results obtained from the 38 cases of bacterial meningitis.{Figure 1}
Table 6: Microbiological fi ndings in CSF samples in meningitis

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Table 7: Age-wise distribution of aetiological agents of bacterial meningitis (n=39)

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Table 8: Aetiological organisms in bacterial meningitis as detected by different diagnostic tests

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   Discussion Top


Bacterial meningitis plays a major role in morbidity and mortality, despite the availability of effective antibiotics. The diagnosis of bacterial meningitis with the identification of the causal agent plays a crucial role in preventing unnecessary associations or the indiscriminate use of broad-spectrum antibiotics.

The present study observed cases of aseptic meningitis more frequently during spring and early fall, whereas cases of bacterial meningitis during the winter and spring seasons where different from other studies who reported ABM in winter and fall with 72.9% and in another only 27.1% cases in spring and summer. [5],[18] These changes were probably because of geographical difference [Figure 1].

In aseptic meningitis, the maximum number of cases was in the age group of 18-65 years, whereas in bacterial meningitis most cases were in the age group of 5-18 years. Previous studies reported that of the patients who had bacterial meningitis, 79% were older than 15 years and 45% were older than 2 years. [19] In the present study however, 68.4% of the cases who had bacterial meningitis were above 5 years of age. This was probably because in the present study there were lesser isolations of pathogens, such as S. agalactiae, Listeria monocytogens, and H. influenza, causing meningitis in neonates and early childhood. In addition majority of the patients inducted in our study belonged to the age group of 5-65 years (56 patients) compared with the age group of 0-5 years (27 patients). Males were found to be affected more commonly. The overall male to female ratio was found to be 1.51:1, which was similar to other studies, with a ratio of 1.2:1 [5],[20] and 1.69:1. [18]

Fever was a predominant finding in all our cases, which was consistent with other studies. [19],[21],[22] Around 75.9% of patients in this study had altered sensorium, which was different from other reports with only 9.1%. [22] Neck rigidity could be elicited in a small number of cases (only 15.7%), which was lesser than that elicited in another study (36.4%). [21] This could be owing to the fact that their study group comprised of only 12 patients with invasive meningococcal disease [Table 1].

In macroscopic finding, 73.6% of cases of bacterial meningitis showed turbid or cloudy appearance in contrast to only 2.2% of cases of aseptic meningitis, which was similar to another study where they reported 85% of CSF sample with acute bacterial meningitis as turbid [Table 2]. [23]

In bacterial meningitis, a majority of the isolates (60.5%) displayed a cell count ranging from 1001 to 5000/cumm, which was found to be significantly higher statistically compared with aseptic meningitis (P = 0.001) [Table 3]. This is similar to that in another study that reported 65.8% of ABM to have cell counts of more than 1000/cumm. [10] Analysis of CSF glucose levels in cases of bacterial meningitis showed that in the majority of cases (55.3%) CSF glucose levels ranged from 11 to 30 mg/dl, which was found to be significantly lower than that in aseptic meningitis (P = 0.034) [Table 4]. CSF protein levels were found to be between 101 and 250 mg/dl in 71.1% cases, which was also found to be significantly higher than that in aseptic meningitis (P = 0.000). None of the CSF samples tested had protein levels less than 50 mg/dl [Table 5].

Smear positive cases and culture were 27.7% and 45.8% higher respectively, than those in previous reports [10],[21] which denotes that culture remains the gold standard technique [Table 6]. The most common isolate was Escherichia coli (25.6%), followed by Staph. aureus (15.4%), Pseudomonas areuginosa, Klebsiella sp., and Strep. pneumoniae were detected in (10.3%) cases each. Citrobacter sp., Providencia sp., and M. tuberculosis where detected in 1 case each [Table 7], [Figure 2]. These findings were different from those in other studies, [20] probably because this study included all age groups unlike the other studies, which included only children.
Figure 2: Positive percentage of bacterial isolates in CSF culture

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We were also able to examine the relative frequency of meningitis caused by E. coli followed by Streptococcus agalacteae in the age group of patients older than 6 months, unlike in previous studies that reported Strep. pneumoniae to be the most common isolate. [10] This was probably because the other studies included subjects with community-acquired meningitis and therefore the number of Gram-negative isolates were comparatively lower than those of the present study. In a study conducted in Nepal, ABM was found in 8.3% cases, the causative agents being Pseudomonas areuginosa (24.3%), coagulase negative Staphylococcus (CONS) (10.8%), and E. coli (2.7%), where they claimed to have hospital-acquired infection causing meningitis. These findings were quite similar to the current study with respect to the organism, more precisely Gram-negative isolates. [24] Blood cultures were positive in only 56.6% cases of bacterial meningitis, which was similar to other findings with 58.4% positivity [Table 6]. [20]

LAT test detected bacterial antigens in 16.8% cases of bacterial meningitis, with the most common organism being E. coli (71.4%), followed by Strep. pneumoniae (28.5%). This finding was similar to that in other studies, where LAT was positive in 15.4% cases of bacterial meningitis. [25] No CSF specimen was positive for N. meningitidis B/C/Y/W/35 and H. influenzae type b [Table 8].

Thus to conclude, the findings in this study show a preponderance of Gram-negative enteric bacilli as the causative agent of bacterial meningitis. H. influenzae and N. meningitidis were not isolated despite all our efforts. This could probably be due to the wide reach of Hib vaccination for H. influenzae in children even in rural and semi-urban areas. N. meningitidis meningitis, on the other hand, is known to occur in epidemics and isolation rates during inter-epidemic periods are generally low. These two organisms are also very delicate and die easily. Tests to detect their antigen in CSF have been more fruitful. Isolations of Gram-positive bacteria were comparatively lower in number. The organisms isolated were Staph. aureus, Strep. pneumoniae, CONS, and S. agalactiae. Further studies however, regarding the occurrence of other pathogens causing bacterial meningitis such as H. influenzae, N. meningitidis, Listeria monocytogenes, and fungi such as Cryptococcus neoformans are required in the future, preferably using tests to detect antigens and even by polymerase chain reaction of the CSF especially considering the fact that these organisms, which are established causes of meningitis were not isolated in the present study.

 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]


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